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The present scope of Liquid Biomarkers in Diagnosis and Prognosis of ovarian cancer- an individualized approach

Professor Jayanta Chatterjee FRCOG, FICP, DFRSH, PhD (London)

Professor in Gynaeoncology, Brunel Medical School, Brunel University, London

Consultant in Gynae-oncology and Gynaecology

Clinical Director in Gynae-oncology, Department of Gynae-oncology, Royal Surrey Foundation Trust hospital

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Ovarian cancer- a heterogeneous disease

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Pathophysiology of High grade serous ovarian cancer

Pathophysiology of high grade serous ovarian cancer

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Mortality Rate

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The 20 Most Common Causes of Cancer Deaths: 2007-2009 and 2017-2019

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Types of liquid biopsies

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Liquid Biopsy vs Tissue Biopsy

Lone et al. Mol Cancer 21, 79 (2022)

Clinical trials ongoing

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CTCs can survive blood stream pressures, and facilitate metastases development

Chemi et al., Front. Oncol. 11 (2021)

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Use of liquid biopsies in our laboratory

www.cbcel.org

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Potential prognostic role of CCs in ovarian cancer

Kumar et al., 2019 Cells

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Identification of CTCs in ovarian cancer

Brightfield PanCK DRAQ5

Kumar et al., 2019 Cells

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CTCs in Clinical Practice

Adapted from Xiao et al. Biomedicines. 9, 9 (2021)

Study	Study Population	Study Treatment	CTC Measurement	Results
Punnoose et al., 2012	Advanced NSCLC	Erlotinib + Pertuzumab	CTC enumeration, EGFR expression in CTCs, oncogenic mutations in CTCs	-Higher baseline CTC counts were associated with treatment response -Larger CTC count decreases at days 14, 28, 56 associated with treatment response
Ignatiadis et al., 2018	High risk, HER2 nonamplified, early breast cancer	Trastuzumab	CTC enumeration at baseline and week 18	-Trastuzumab did not decrease rate of CTC detection
Tan et al., 2018	Colorectal	Chemotherapy (broad)	CTC enumeration	CTC count trends can be used in place of uninformative CEA for monitoring patient response
Bonvini et al., 2021	Inflammatory myofibroblastic tumor	Entrectinib	Longitudinal CTC enumeration during treatment (up to 24 months post treatment)	Antitumor activity was associated with elimination of CTCs from blood
Sperger et al., 2021	Metastatic prostate cancer	Enzalutamide or abiraterone	CTC androgen-receptor (AR) gene expression	-CTCs clustered according to AR-gene expression -Presence of CTCs enriched for AR-gene transcripts associated with shorter OS
Pang et al., 2021	Metastatic Breast Cancer	Surgery or Adjuvant Therapy	CTC enumeration	CTC positivity associated with shorter OS and PFS following adjuvant therapy

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Current Clinical Trials (CTCs) – Recruiting status (21/10/24)

784

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The CTC market is projected to grow from

$10.7 billion in 2022 to $30.7 billion by 2029

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Circulating Tumour DNA (ctDNA)

ctDNA is tumour DNA that has been shed into the bloodstream

ctDNA can be present in 0.01% - >90% of the total cell free DNA (cfDNA)

The amount of ctDNA is related to the tumour burden and varies between patients with different clinical presentations

Diaz & Bardelli,. J Clin Oncol. 32,579 (2014)

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Companion Diagnostic Labels for

Guardant360 CDx

	Biomarker	Therapy
Lung cancer NSCLC	EGFR exon 19 deletions,	TAGRISSO®
	L858R, and T790M†	(osimertinib)
	EGFR exon 20 insertions	RYBREVANT®
	EGFR exon 20 insertions	(amivantamab-vmjw)
	ERBB2/HER2 activating mutations	ENHERTU®
	(SNVs and exon 20 insertions)	(fam-trastuzumab deruxtecan-nxki)
	KRAS G12C	LUMAKRAS™
	KRAS G12C	(sotorasib)
	Biomarker	Therapy
Breast cancer	ESR1 missense mutations	ORSERDU™
	between codons 310-547	(elacestrant)

www.guardantcomplete.com

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New findings in ovarian cancer relating to the PD-1/PD-L1 pathway

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Fig. 1 PD-L1 expression on monocytes correlates with malignancy and is upregulated by ascitic supernatant (asc sn). a Percentage of PD-L1 positive cells in the CD14 gate of PBMCs from patients with benign/borderline (n = 29) or malignant ovarian cancer (n = 29). b % of PD-L1 positive cells in the monocyte gate of ascites from patients with benign/borderline (n = 10) or malignant ovarian cancer (n = 44). c Histogram from flow cytometry showing increased PD-L1 cell surface expression on monocytes (gated on CD14 positive cells) isolated from PBMCs from representative patients with a benign or malignant ovarian tumor compared to antibody isotype control samples. d Monocytes (CD14+) were purified from healthy donors and cultured in ascitic supernatant harvested from patients with either

benign (n = 3) or malignant (n = 4) ovarian tumors for 0, 2, 6 and 8 h. The cells were then stained for PD-L1 and analyzed by flow cytometry. The experiments were carried out twice, and the data have

been pooled. The figure shows each group’s mean value of MFIs for each time point ± SEM

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Cytokine regulation of PD-L1 expression on monocytes

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Cytokine regulation of PD-L1 expression on monocytes.

Using a Luminex assay, IL-10 (a) and TGF-β (b) concentrations were measured in the ascitic supernatant of patients with either benign/borderline

(IL-10, n = 8; TGF-β, n = 9) or malignant (IL-10, n = 42; TGF-β, n = 24) ovarian tumors. Each data point represents 1 patient sample and the bar represents the mean c IL-10 or TGF-β can upregulate

PD-L1 on monocytes. Monocytes from PBMCs from healthy individuals were cultured in medium containing IFN-γ, IL-10, TGF-β or IL-10+, TGF-β for 0, 2 and 8 h. PD-L1 expression was

analyzed by flow cytometry and expressed as mean ± SEM of triplicate cultures, and the figure shows a representative experiment of two. d Monocytes from healthy individuals were cultured in medium

or ascitic supernatant (asc sn) from patients with either high IL-10 concentration (>100 ng/ml) or low IL-10 concentration (<35 ng/ml). IL-10 blocking antibody was added into the cultures as indicated

(anti-IL-10 mAb was used at 1 μg/ml. “x5” = 5 μg/ml), and PD-L1 expression was measured after 8 h by flow cytometry. This experiment was performed in triplicate and repeated twice, and the figure

shows the mean ± SEM. *p < 0.05, **p < 0.001

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PD-1 is upregulated on T cells from patients with ovarian cancer

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PD-1 cell surface expression on T cells isolated from PBMCs (a) (B/B n = 23, malignant n = 29) and ascites (b) (B/B n = 12, malignant n = 44) from patients with malignant compared

to benign/borderline ovarian tumors. Graphs represent individual cases of benign/borderline or malignant, while horizontal lines represent mean values. c CFSE-labeled, bead-stimulated T cells (ST)

derived from healthy donors were co-cultured with ascitic monocytes (AM) or monocytes from a healthy female donor; with and without anti-PD-1, anti-PD-L1 and anti-IL-10 blocking antibodies alone or

in combination. Each bar represents the percentage of stimulated T cells that underwent proliferation after 5 days of co-culture. Error bars = SEM. d CFSE-labeled T cells from the ascites of a patient

were co-cultured with monocytes derived from either ascites or the PBMCs of healthy female donor. Each bar represents the mean percentage

of stimulated T cells that underwent proliferation after 48 h of co-culture. Data shown are the mean ± SEM of triplicate cultures

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Clinical use of the pathway as biomarkers

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Non invasive nature
Use of biofluids (blood, urine, sputum, saliva).
Less trauma and generally less risk to patients, especially those who are not candidates for invasive biopsy
Signatures of patients’ tumours can be characterized molecularly; aids in understanding tumor biology
May be the best tool for monitoring therapeutic response and perhaps, early detection.
Repeated longitudinal sampling is possible.
ctDNA might reflect better tumour heterogeneity than tissue biopsies.

Advantages of Liquid Biopsy

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Challenges: Technological, Clinical and Biological

What is the clinically relevant biofluid to sample and from where?

Low levels of ctDNA; thus requiring large volumes of blood

Issues with capture and culture efficiency of CTCs

How truly representative are CTCs of tumour heterogeneity?

What are the disease relevant CTCs (e.g. clusters)?

Is there sample bias? (i.e., are all regions of the tumour the same?)

Is liquid biopsy representative of all the genetic changes of a tumour?

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CBCEL

Dr Sayeh Saravi
Radwa Hablase
Rebecca Karkia
Suzana Panfilov
Anna Migut
Jovile Kazileviciute
Joanne Dias

Alumni

Dr Zena Alizzi
Dr Alice Filipe
Dr Juhi Kumar
Dr Rachel Kerslake
Dr Periklis Katopodis
Dr Aeman Zahra
Rooban Jeyaneethi
Dr Dimple Chudasama
Dr Sophie Jahn
Dr Georgios Sotiriades
Dr Karly-Rai Rogers
Dr Julie Davies
Dr Elena Zachariades

www.cbcel.org